N5-(carboxyethyl)ornithine synthase (EC 1.5.1.24) from Lactococcus lactis subsp. lactis mediates the NADPH-dependent reductive condensation between L-ornithine (or L-lysine) and pyruvate to form N5- (L-1-carboxyethyl)-L-ornithine (or N6-(L-1-carboxyethyl)-L-lysine). To study the relationship between this enzyme and the three other N- (carboxyalkyl)amino acid dehydrogenases that have been sequenced, the gene ceo was cloned (from L. lactis K1), expressed in Escherichia coli, and sequenced. The gene encodes a 313-amino-acid protein (Mr = 35,323) which showed relatively little overall sequence similarity to proteins currently catalogued in the data banks. When compared with the other N- (carboxyalkyl)amino acid dehydrogenases, N5-(carboxyethyl)ornithine synthase was most similar to yeast saccharopine dehydrogenase (EC 1.5.1.7), which catalyzes the NADH-dependent condensation of lysine and alpha-ketoglutarate. These two proteins constitute a family of Nomega- (carboxyalkyl)amino acid dehydrogenases, which in turn, can be considered part of an amino acid dehydrogenase superfamily, that would also include N2-(carboxyalkyl)amino acid dehydrogenases such as octopine synthase and nopaline synthase. With respect to the binding of its three substrates, N5-(carboxyethyl)ornithine synthase contains a sequence segment that was virtually identical (eight out of nine residues) to one present in the betaalphabeta-fold of the dinucleotide- binding domain of several microbial glutamate dehydrogenases. In addition, sequence motifs associated with the binding of pyruvate and ornithine were identified in the N-terminus of the enzyme.